Longevous cultures of aciduric bacteria



Patented July 11, 1933 UNITED STATES FERDINAND W. NITAR-DY, OF BROOKLYN,

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NEW YORK, ANID'JOHN J. 'ENBIGH'J, AND

VINCENT S. WRENN, PITTSBURGH, PENNSYLVANIA, ASSIGNORS TO E. B. SQUIBB & SONS, OF NEW YORK, N. Y., A CORPORATIONOF NEW YORK LONGEVOUS CULTURES OF ACIDUBIC BACTERIA No Drawing. Application fi1ed April 1,

Our inventionrelates to cultures of aciduric bacteria. 7 a

It has for some years been recognized by the medical profession that the implantation and proliferation of aciduric bacteria-for marketed for accomplishing this implantation have been characterized by the early death of the major part of their bacilli, owing to the high concentration of lactic acid produced by them. Even in milk, authoritatively held to be the best medium, the microorganismsare decimated with such rapidity that their count is reduced to a small per centage in a few days. As a consequence of this instability, a. considerable economic Waste has been suffered; and daily distribution of fresh cultures has been rendered necessary, wherefore preparations of the fullest therapeutic value can be supplied only to communities situated within certain territorial limits. Moreover, the milk medium commonly employed for cultures is quickly soured by the metabolic processes of the bacteria, and thus rendered extremely unpalatable to many who would otherwise avail themselves of the treatment. Broth cultures flavored with fruit juices are known, but ther bacteria are so ephemeral that such cultures have to be consumed in volumes about thirty times as great as those of fresh 1in; cultures to obtain the same effective ose.

It is an object of our invention to provide longer-lived cultures of aciduric bacteria than those hitherto known. A further object is to provide such longevous cultures optionally in media, of unobjectionable taste, other than milk. to provide a method whereby these cultures may be prepared. 7 i

In the practice of our invention we maintain the cultures of aciduric bacteria in the presence of a growth-inhibiting agentby A still further object is 1931. semi in. 527,040.

which term we mean to include all substances 1 4 tnat inhiblt or reduce bacterial growth without impairing the viability of the micro-organisms; and thus, by completely or partially suspending the vital activities of the bacteria and keeping them in arelatively dormantstate, prevent them from generatlng products by Which theywouldbe killed. We have found, for instance, that the appli catlonof carbon dioxide (which may be ininteraction of suitable acids and carbonates) in a concentration'of betweenone and five, optimally for ordinary purposes aboutztwo, volumes-will accomplish our purpose.

I j 00' troduced as such or generated in situ by the A specific example of the practice of our invention is as follows: A peptoneovhey broth medium is first prepared by heating skimmed milk to between ,and (I, enough ten-percent hydrochloric acid is add separated by filtration through several thicknesses of cheese-cloth, and the reaction being adjusted to pHiiO with ten-percent sodium hydroxide, the whey is placed in flasks v ,70 ed to precipitate all the casein, the whey is" plugged with cotton, which are autoclaved at twenty iounds for thirty minutes: the

iactalbumin settles, and after the supernatant whey is decanted and filtered, 5.0 grams of peptone is added to the liter of whey, the reaction is adjusted to pH 6.0, and sterilization is effected by autoclavin Selection is made of an actively growing; strain of Lactobacilius aoiclophe'lus of the desired type, which is passed through several succes sive transplants in sterile peptone-whey broth until a heavy seeding culture can be obtained by three or four days incubation,

when 100 cc. is transferred to the large flask containing; one liter of sterile peptone-wliey broth; and incubated at 37 C. for 72 hours. Then 25 cc. quantities of the culture are aseptically transferred to sterile 200 cc. bottles containing, sterile, 40 cc. of 27.7" Baum sucrose syrup and 8 cc. of true-fruit strawberry extract; and the bottles are filled with" carbonated water of such gaspressure that the final concentration of carbon dioxide in the sealed product will be approximately two Volumes.

By the word cultures herein, We mean to include all potable and edible preparations containing live micro-organisms, for examplein the formof liquids, pastes,gelatinizedimassesor moist solid-s.

It is to be understood that the foregoing example is merely illustrative and by no means definitive of our invention, which within the scope of the appended claimsmay embody various types of micro-organisms, culture media (including milk), growth-inhibiting agents, and processes, and may be employed in various industrial fermentative operations.

We claim:

.11. The method of prolonging the life of cultures of Lactobacillus which comprises inaintainlngithem 1n the presence ofcarbon dioxide .in a concentration of between one :andsfive volumes.

'2- Theunethod of prolongingthedife of cultures. ofiLactobacillus which comprisesmain- .taining them in the-presence ofcarbon dioxide ,in a concentration of about two volumes.

3. The method of prolonging the lifeof cultures of lactoba-o'illu s ao'izlophilus which comprises maintaining them in the presence 7 In\ivitnesswhereof weai'fix our of carbon dioxide in a concentration of be tween one and five volumes.

4. The method of prolonging the life of lofabout two volumes.

. .9. Culturesor" Lactobacillue acidophflm, in nnedia other than milk, including carbon dioxide in a concentration ofnhouttwo volumes.

gnaturos. FERDINAND WV. Nl FAQXDY. JOHNIJ. ENRIGHT.

VINCENT S. RENN. 

